The long term goals of our work are to understand how epithelial cells organize into monolayers through specialized cadherin-mediated cell-cell contacts, and localize proteins to functionally different plasma membrane domains. We integrate innovative experimental approaches to address these problems: structural analysis of proteins and protein complexes, high resolution live cell imaging of proteins and biosensors, biochemical analysis of protein complex assembly and function in cells, and novel in vitro reconstitution assays. During this funding period, we defined stages in cell-cell adhesion and the mechanism involved, and determined how plasma membrane proteins are targeted to and organized in the forming basolateral membrane domain upon cell-cell adhesion. We will build upon these results in proposed studies. We will define roles of alpha-catenin and p120 in regulating cell-cell adhesion and membrane dynamics. We will examine the effects of sequestering endogenous alpha-catenin and p120 to the plasma membrane independently of E-cadherin, and to mitochondria using location-specific tags on cell migration, actin dynamics and cell-cell adhesion using live cell imaging, FRET, biosensors and small molecule inhibitors. We will use affinity purification and MALDI/TOF Mass Spectrometry to define proteins that regulate alpha-catenin and p120 functions and association with the actin cytoskeleton. We will dissect mechanisms regulating sorting of vesicles at the basolateral plasma membrane targeting patch. We will use innovative reconstitution assays on membrane patches bound to an E-cadherin substrate to visualize how transport vesicles are delivered to the targeting patch, and how each of the componentsof the targetign patch function. These studies will be extended to functions of Lgl, Discs Large, Scribble and Par proteins in the development of cell surface polarity. We will investigate the role of microtubule (MT) and septin cytoskeleton in cell-cell adhesion and vesicle delivery to the basolateral plasma membrane targeting patch. We will define roles of septins in cell-cell adhesion and in specifying Glu-MT organization and vesicle trafficking towards the plasma membrane, and investigate protein-protein interactions between septins/MTs and the targeting patch. Together, these studies will provide a comprehensive molecular picture of the signaling and structural protein networks involved in cell-cell adhesion and the development of cell surface polarity.